Distribution of penicillin in the cornea, tears and aqueous humour at various times following a single intravenous injection

Bioassays performed by the disc diffusion technique on samples of blood, tears, aqueous humour and cornea from 30 rabbits killed 4 to 24 hours following a single intravenous injection of benzylpenicillin (40 mg/kg) showed that the penicillin content of these fluids and tissues progressively decreased with time. However, even after 24 hours the agent could be detected in the samples. The peripheral and central cornea contained similar amounts of penicillin up to 16 hours; thereafter, the peripheral cornea contained more of the agent.     

Application of a U-13C-labeled amino acid tracer in lactating dairy goats for simultaneous measurements of the flux of amino acids in plasma and the partition of amino acids to the mammary gland

A preliminary study was conducted using lactating British Saanen goats (n = 5) at 109 to 213 d in milk that yielded 1.67 to 3.68 kg of milk/d to examine the application of a U-13C-labeled amino acid (AA) mixture obtained from hydrolyzed algal proteins as a tracer for measuring plasma flux (n = 5) and partition to the mammary gland (n = 3; arteriovenous difference) of 13 AA simultaneously. Except for Ile and Ser, there was incomplete (6 to 54%) equilibration of the tracer with AA from packed blood cells (> 90% erythrocytes) during the 6-h infusions. This result agreed with the large ratio of packed cells to gradients for plasma AA concentration that was also observed. However, net mass and isotope removals by the mammary gland were predominantly from plasma, indicating that the erythrocytes did not participate in kinetic exchanges. Plasma AA fluxes (millimoles per kilogram of metabolizable protein intake per kilogram of body weight 0.75) differed among goats that consumed different protein sources; however, overall rates were lowest for Met (5 to 14) and His (8 to 17) and highest for Leu (48 to 70) and Ala (53 to 88). On average, 25% of plasma flux was partitioned to the mammary gland. Less than 20% of His, Ser, Phe, and Ala were directed to the mammary gland; 20 to 30% of Arg, Thr, Tyr, and Leu were directed to the mammary gland; and 30 to 40% of Pro, Ile, Lys, and Val were directed to the mammary gland. The unidirectional AA flux in the mammary gland (AA apparently available for protein syntheses, oxidation, and metabolite formation) did not match the pattern that is required for casein synthesis, suggesting differences in the metabolic requirements of AA for nonmilk protein synthesis.     

Effect of the non-NMDA receptor antagonist GYKI 52466 on the microdialysate and tissue concentrations of amino acids following transient forebrain ischaemia

The effect of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) on ischaemia-induced changes in the microdialysate and tissue concentrations of glutamate, aspartate, and gamma-aminobutyric acid (GABA) was studied in rats. Twenty minutes of four-vessel occlusion resulted in a transient increase in microdialysate levels of glutamate, aspartate, and GABA in striatum, cortex, and hippocampus. Administration of GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min intravenously starting 20 min before onset of ischaemia) inhibited ischaemia-induced increases in microdialysate glutamate and GABA in striatum without affecting the increases in hippocampus or cortex. Twenty minutes of four-vessel occlusion resulted in immediate small decreases and larger delayed (72 h) decreases in tissue levels of glutamate and aspartate. Transient increases in tissue levels of GABA were shown in all three structures at the end of the ischaemic period. At 72 h, after the ischaemic period, significantly reduced GABA levels were observed in striatum and hippocampus. GYKI 52466, given under identical conditions as above, augmented the ischaemia-induced decrease in striatal tissue levels of glutamate and aspartate, without significantly affecting the decreases in hippocampus and cortex. Twenty minutes of ischaemia resulted in a large increase in microdialysate dopamine in striatum. GYKI 52466 failed to inhibit this increase. Kainic acid (500 microM infused through the probe for 20 min) caused increases in microdialysate glutamate and aspartate in the striatum. GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min) completely inhibited the kainic acid-induced glutamate release. In conclusion, the action of the non-NMDA antagonist, GYKI 52466, in the striatum is different from that in the cortex and hippocampus. The inhibition by GYKI 52466 of ischaemia-induced and kainate-induced increases in microdialysate glutamate concentration in the striatum may be related to the neuroprotection provided by GYKI 52466 in this region.     

In vitro incorporation of GPI-anchored proteins into human erythrocytes and their fate in the membrane

In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4 degrees C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4 degrees C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.     

Distribution of free D-amino acids in tissues and body fluids of vertebrates

Reports on the distribution, metabolism and origins of free D-amino acids in vertebrate tissues and body fluids are reviewed. The transient emergence of D-aspartic acid during the development of brain and peripheral organs or early stages of life is reported. D-Serine in brain is postulated to be a potentiator for the N-methyl-D-aspartate (NMDA) receptor. Some D-amino acid concentrations in human serum, such as D-Ser and D-Ala, are suggested to correlate with damage to renal function.     

The intake of proteins and essential amino acids in top-competing women athletes]

PURPOSE: The literature on diagnostic peritoneal lavage in the assessment of blunt abdominal trauma reflects an ongoing controversy. Therefore we conducted a prospective evaluation of the diagnostic management of blunt abdominal trauma used at our clinic, in which this procedure plays a substantial role. During the years 1993 and 1994 a total of 75 patients could be included in the study. The study population consisted of all patients with a diagnosis of blunt abdominal trauma. In addition, all trauma patients who were unresponsive on admission to the emergency receiving unit underwent the same program of diagnostic work-up. This group included polytraumatized patients, patients with craniocerebral injuries and all those who had been intubated prior to admission. Patients with stable vital signs were evaluated first by sonography of the abdomen, whereas those showing signs of hypovolemic shock received a diagnostic peritoneal lavage as the first evaluation of abdominal trauma. In order to assess the relative value of the two diagnostic methods, all patients who had had ultrasound as their first examination subsequently also underwent peritoneal lavage. RESULTS: 37 patients (49%) had lavage evidence of intraperitoneal bleeding. Of these 22 (29% of the total) subsequently underwent emergency laparotomy with lesions requiring surgical treatment found in 21 (95%). Only in one patient (1.3% of the study population) laparotomy failed to reveal a lesion requiring surgical correction. The accuracy of peritoneal lavage findings as an indication for laparotomy was 99%, compared to 82% for ultrasonography used as a initial diagnostic procedure. Diagnostic peritoneal lavage is quick, safe and almost independent of the experience of the investigating physician. It can be performed during other diagnostic procedures and can be repeated at will. If beyond macroscopical evaluation the lavage fluid is assessed chemically, even duodenal and pancreatic lesions as well as injuries to other hollow viscera can be suspected. With a sensitivity of 100% and a specificity of 98%, diagnostic peritoneal lavage is an extremely reliable diagnostic tool. It should be used as the initial diagnostic procedure in all hypovolemic and/or unresponsive patients suspected of having suffered blunt abdominal trauma. In conscious patients with stable vital signs, ultrasonography can be used for initial diagnosis. It should, however, be complemented by subsequent peritoneal lavage whenever the clinical course gives rise to suspicion.     

In vivo incorporation of glucose carbon into brain and liver proteins in biotin-deficient rats

The in vivo utilization of D[U-14C]glucose with particular reference to its incorporation into brain and liver proteins was studied in biotin-deficient rats. The data show a significant reduction in the incorporation of glucose carbon into brain and liver proteins. A disturbance in the intermediary metabolism of glucose to be one of the factors responsible for the decreased incorporation of the label into proteins in biotin deficiency.     

Effects of human chorionic gonadotropin preparations on amino acid uptake and incorporation into protein in vitro

Human chorionic gonadotropin preparations were tested for their effect on protein metabolism by cultured fibroblasts. Commercial preparations of HCG strongly inhibit amino acid uptake and incorporation into protein by a variety of human adult and fetal fibroblast lines, whereas, highly purified HCG is without such an effect.     

Kinetics of the incorporation of dietary fatty acids into serum cholesteryl esters, erythrocyte membranes, and adipose tissue: an 18-month controlled study

Tissue levels of n-3 fatty acids reflect dietary intake, but quantitative data about rate of incorporation and levels as a function of intake are scarce. We fed 58 men 0, 3, 6, or 9 g/d of fish oil for 12 months and monitored fatty acids in serum cholesteryl esters, erythrocytes, and subcutaneous fat during and after supplementation. Eicosapentaenoic acid (EPA) in cholesteryl esters plateaued after 4-8 weeks; the incorporation half-life was 4.8 days. Steady-state levels increased by 3.9 +/- 0.3 mass % points (+/- SE) for each extra gram of EPA eaten per day. Incorporation of docosahexaenoic acid (DHA) was erratic; plateau values were 1.1 +/- 0.1 mass % higher for every g/d ingested. Incorporation of EPA into erythrocyte membranes showed a half-life of 28 days; a steady state was reached after 180 days. Each g/d increased levels by 2.1 +/- 0.1 mass %. C22:5n-3 levels increased markedly. Changes in DHA were erratic and smaller. EPA levels in adipose tissue rose also; the change after 6 months was 67% of that after 12 months in gluteal and 75% in abdominal fat. After 12 months each gram per day caused an 0.11 +/- 0.01 mass % rise in gluteal fat for EPA, 0.53 +/- 0.07 for C22:5n-3, and 0.14 +/- 0.03 for DHA. Thus, different (n-3) fatty acids were incorporated with different efficiencies, possibly because of interconversions or different affinities of the enzymatic pathways involved. EPA levels in cholesteryl esters reflect intake over the past week or two, erythrocytes over the past month or two, and adipose tissue over a period of years. These findings may help in assessing the intake of (n-3) fatty acids in epidemiological studies.     

Amino acid incorporation into liver and tumor proteins in patients with hepatic neoplasms-effects of liver dearterialization

The effects of liver dearterialization on the rate of amino acid incorporation into liver and tumor proteins were studied with an in vitro method in seven patients with liver metastases. Before liver dearterialization the incorporation rate was 0.074 +/- 0.020 nmol leucine x mg prot-1 x h-1 in liver tissue and 0.234 +/- 0.049 nmol leucine x mg prot-1 x h-1 in tumor tissue. After dearterialization for 1 h the incorporation rate was reduced to about half of the initial values in both liver and tumor tissue. The vascularity of the tumors was evaluated from preoperative hepatic angiograms. The reduction of the incorporation rate was more pronounced in highly vascularized tumors than in poorly vascularized tumors and liver tissue. The clinical implications of a more pronounced metabolic effect of the dearterialization in high vascularized tumors are discussed.     

Characterization of azo coupling adducts of benzenediazonium ions with aromatic amino acids in peptides and proteins

A synthetic peptide, VLSPADKTNWGHEYRMF(cmC)QIG, was reacted with 4-chlorobenzenediazonium hexafluorophosphate as a model for reactions of aromatic diazonium ions with proteins. At a ratio of diazonium ion to peptide of 0.8:1, three products could be seen by reversed-phase HPLC. Electrospray mass spectrometric analysis of the isolated products revealed that two of the products had the same mass of 2648 Da, being 138 Da higher than the parent peptide and corresponding to the addition of a 4-chlorobenzenediazo group. The third isolated product had a mass of 2787 Da which corresponded to the addition of two 4-chlorobenzenediazo groups (276 Da). Digestion of the monoadducted intact peptides with trypsin or endoproteinase Glu-C and HPLC separation of adduct oligopeptides followed by sequencing with electrospray ionization tandem mass spectrometry showed unambiguously that histidine and tyrosine residues were the major sites of modification. Incubation of human serum albumin with 4-chlorobenzenediazonium hexafluorophosphate at molar ratios of 1:1, 1:2, and 1:10 resulted in adduct formation as detected by shifts in the HPLC retention time of the protein and also by an increase in mass as determined by electrospray mass spectrometry.     

Effect of free amino acids and vitamins on cleavage and developmental rate of bovine zygotes in vitro

Due to the complicated media used for culturing bovine embryos, most of the nutrient requirements are unknown. Recently, we developed a simple, serum-free medium (CR1) that allows bovine embryos to develop in vitro. Therefore, our objective was to determine whether development of bovine embryos would be improved by the addition of free amino acids and vitamins to CR1. Oocytes were recovered from slaughterhouse ovaries and matured 22 +/- 2 h, following which the oocytes were randomly allotted to treatment. The experiment was a randomized block design with a 2 x 5 factorial treatment structure. The oocytes were fertilized with or without cumulus cells intact. The five fertilization media were 1) Control (CR1 +/- 10 micrograms/mL of phenol red); 2) control + basal medium Eagle (BME) essential amino acids (EAA) + minimum essential medium (MEM) nonessential amino acids (NEA) + MEM vitamins (VIT); 3) control + EAA + NEA; 4) control + EAA + VIT; and 5) control + NEA + VIT. Cleavage rate was greater (P < .001) when cumulus cells remained on the oocytes during fertilization (51.7 vs 73.2% without and with cumulus cells, respectively). The frequency of blastocysts was increased (P < .001) when EAA or NEA were added to CR1; however, adding VIT had no effect or tended (P = .12) to decrease the frequency of embryos attaining the blastocyst stage. This experiment demonstrates that development of bovine embryos in vitro can be improved by the addition of free amino acids to a simple medium. Contrary to work in rodents, the mixture of vitamins in MEM was not beneficial for bovine embryos.     

Fluorescence detection of proteins and amino acids in capillary electrophoresis using a post-column sheath flow reactor

A simple laser-induced fluorescence detection method for proteins and amino acids in capillary electrophoresis is reported. A sheath flow cell is utilized as a post-column reactor for fluorescence derivatization of proteins and amino acids by addition of o-phthaldialdehyde-2-mercaptoethanol to the sheath fluid. With the use of a 50 microns I.D. capillary, the limits of detection for carbonic anhydrase are 0.73 nM or 1.8 amol which represents a five- and two-fold improvement, respectively, over the best results previously reported for post-column detection. In addition, separation efficiencies up to 8.07 x 10(5) are achieved and the detector response is linear over three-orders of magnitude. These results demonstrate that mixing is adequate and the reaction kinetics are rapid enough to provide sensitive detection with this approach. Also, because this post-column derivatization scheme requires no instrumental changes to a typical sheath flow cell detector, the system can be used for detection of pre-column labeled analytes and for native fluorescence detection.     

Assigning the NMR spectra of aromatic amino acids in proteins: analysis of two Ets pointed domains

The measurement of interproton nuclear Overhauser enhancements (NOEs) and dihedral angle restraints of aromatic amino acids is a critical step towards determining the structure of a protein. The complete assignment of the resonances from aromatic rings and the subsequent resolution and identification of their associated NOEs, however, can be a difficult task. Shown here is a strategy for assigning the 1H, 13C, and 15N signals from the aromatic side chains of histidine, tryptophan, tyrosine, and phenylalanine using a suite of homo- and hetero-nuclear scalar and NOE correlation experiments, as well as selective deuterium isotope labelling. In addition, a comparison of NOE information obtained from homonuclear NOE spectroscopy (NOESY) and 13C-edited NOESY-heteronuclear single quantum correlation experiments indicates that high-resolution homonuclear two-dimensional NOESY spectra of selectively deuterated proteins are invaluable for obtaining distance restraints to the aromatic residues.